Alpha-amylase assay and uses thereof

ABSTRACT

This invention relates to methods and kits for measuring α-amylase activity in samples such as flour, stock, or amylase concentrate.

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.10/230,969, filed Aug. 29, 2002, now pending.

FIELD OF THE INVENTION

This invention relates to methods and kits for measuring α-amylaseactivity in grain products such as flour.

BACKGROUND OF THE INVENTION

The use of flour produced through the processing of cereal grains suchas wheat, rye, and oats is an important feature in nutrition and foodproduction around the world. Grains are grown, harvested, and milledinto flours, which are used to make breads, bakery products, pastas, allof which are staples in the diet of many individuals world wide. Grainsand grain products are also utilized for brewing and fermentation.

Wheat flour is an important ingredient in home baking and is thefoundation for almost every commercially baked product and pasta. Of thegrains available for the production of flour, wheat is unique in that itis the only cereal grain with sufficient gluten content to make a loafof bread without being mixed with another grain. Wheat is grown all overthe world and is the most widely distributed cereal grain. In general, areference to “flour” is a reference to wheat flour. Flour is usedextensively in the food industry and a key requirement in that industryis the uniform high quality and performance of flour and grains in foodand beverage production (For review see: Plant Foods for HumanNutrition, Vol 55:1-86, 2000).

Cereal grains store energy as starch, and to perform well in baking andfood production, it is important to optimize the level of starch inflour. A key factor in the breakdown of starch in flours is the presenceof α-amylase activity in cereal grain flours. α-amylase is an endoenzymethat is present in cereal grains and breaks the α-1,4, glucosidic bondsthat are present in starch. The enzyme works in an almost random mannerand the effect of its enzymatic activity is the breakdown in the size ofthe starch molecules and the conversion of starch to sugars anddextrins.

To help ensure efficient food production methods, it is important to beable to accurately assess the level of α-amylase activity in batches offlour. The presence of excess α-amylase activity flour results in areduction in the value of the flour for baking. For example, excessstarch breakdown in flour can result in sticky or doughy bread thatcan't be cut in automated loaf-slicing machinery and is thereforeunsuitable for commercial production. Insufficient α-amylase activity inflour can also reduce the value of a flour for baking and foodproduction. Insufficient α-amylase activity in flour can result flourthat lacks the necessary levels of sugars for proper fermentation andyeast activity in baking. Flours with insufficient α-amylase arefrequently supplemented with amylase concentrates. Because of thefinancial importance of flour quality in the baking and food productionindustries, it is important to have reliable, reproducible, andeasy-to-use methods to determine the amount of α-amylase activity inflours.

Current methods to determine the level of α-amylase in flour includetechniques such as the Hagberg-Perten Falling Number test. This is aviscosity-based method in which a flour suspension is heated togelatinize the starch. The viscosity of the mixture is determined byputting the suspension into a long narrow tube of defined dimensions andmeasuring the rate at which various calibrated small stirrers or a rodfalls though the suspension in the tube. Although the Falling Numbertest is currently accepted as the industry standard, it does not measurethe actual α-amylase enzyme activity level directly, and it is theactivity of the enzyme that affects baked good texture and value.

Alternative methods that directly measure α-amylase enzyme activity havebeen developed, but are not used to fluorometrically test flour, amylaseconcentrate, or stock samples, which limits their usefulness. Theavailability of a fluorometric method to directly determine α-amylaseenzyme activity in flour, amylase concentrates, or stock samples wouldprovide a more accurate prediction of a flour's, concentrate's, orstock's performance, and therefore its value in the baking industry andin other food and beverage production industries.

SUMMARY OF THE INVENTION

The invention is based, in part, on our surprising discovery that thelevel of α-amylase enzyme activity in a flour sample and/or amylaseconcentrate sample can be determined by contacting a sample from a grainflour or other grain or plant product, or an amylase concentrate sample,with a detectably labeled starch substrate and determining the amount ofhydrolysis of the substrate as a measure of the α-amylase enzymeactivity in the sample.

According to one aspect of the invention, methods for measuringα-amylase activity in a sample are provided. The methods include forminga reaction mixture by contacting a sample with a detectably labeledstarch substrate for a time sufficient for α-amylase in the sample tohydrolyze the starch substrate, thereby releasing soluble detectablylabeled starch fragments, separating the soluble detectably labeledstarch fragments from the reaction mixture, and determining the level ofhydrolysis of the detectably labeled starch substrate as a measurementof α-amylase activity in the flour or stock sample. In some embodiments,the sample is a flour sample. In certain embodiments, the sample is astock sample. In some embodiments, the sample is an amylase concentratesample.

In some embodiments, determining the level of hydrolysis of thedetectably labeled starch substrate includes quantifying the detectablylabeled starch substrate. In other embodiments, determining the level ofhydrolysis of the detectably labeled starch substrate includesquantifying the soluble detectably labeled starch substrate fragments.In some embodiments, the method also includes calculating the α-amylaseactivity in the sample by correlating the quantity of detectably labeledstarch to an α-amylase standard. In some embodiments, the method alsoincludes calculating the α-amylase activity in the sample by correlatingthe quantity of soluble detectably labeled starch fragments to anα-amylase standard. In certain embodiments, the detectably labeledstarch substrate is a potato starch. In certain embodiments, thedetectably labeled starch substrate includes D-glucose residues and islabeled on about one of every 300-1300 D-glucose residues of the starchsubstrate. In some embodiments, the starch substrate is detectablylabeled with a label compound selected from the group consisting offluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent,and bioluminescent molecules. In some embodiments, the label is afluorophore. In certain embodiments, the fluorophore is selected fromthe group consisting of 5-((2-aminoethyl)amino)naphthalene-1-sulfonicacid (EDANS), fluorescein isothiocyanate (FITC), and Marina Blue.

In some embodiments, the step of separating the soluble detectablylabeled starch fragments from the reaction mixture includes filteringthe reaction mixture to remove from the mixture detectably labeledstarch substrate. In some embodiments, the step of filtering includesthe addition of a filtration aid selected from the group consisting ofresin, glass beads, beads, and celite. In some embodiments, the step ofseparating the soluble detectably labeled starch fragments from thereaction mixture includes centrifuging the reaction mixture to removefrom the mixture detectably labeled starch substrate. In someembodiments, the method also includes measuring an aliquot of thesupernatant of the centrifuged reaction mixture. In certain embodiments,the step of separating the soluble detectably labeled starch fragmentsfrom the reaction mixture includes obtaining an aliquot of the reactionmixture and centrifuging the aliquot of the reaction mixture to removefrom the aliquot detectably labeled starch substrate. In someembodiments, the step of separating the soluble detectably labeledstarch fragments from the reaction mixture includes contacting thefragments with an agent that binds to the detectably labeled starchfragments. In certain embodiments, the agent is a lectin. In otherembodiments, the agent is an antibody.

In some embodiments, the sample is an aqueous slurry. In someembodiments, the sample is contacted with the detectably labeled starchsubstrate for a reaction time of at least about 1 sec, 5 sec, 10 sec, 15sec, 20 sec, 25 sec, 30 sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 1min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20min, 21 min. 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56min, 57 min, 58 min, 59 min, or 60 min. Preferably, the sample iscontacted with the detectably labeled starch substrate for a reactiontime at least about 1 minute, at least about 5 minutes, at least about10 minutes, or at least about 15 minutes.

According to another aspect of the invention, methods for measuringα-amylase activity in a sample are provided. The methods include forminga reaction mixture by contacting a sample with a detectably labeledstarch substrate attached to a surface, for a time sufficient forα-amylase in the sample to hydrolyze the starch substrate, therebyreleasing soluble detectably labeled starch fragments, separating thesoluble detectably labeled starch fragments from the reaction mixture,and determining the level of hydrolysis of the detectably labeled starchsubstrate as a measurement of α-amylase activity in the sample. In someembodiments, the sample is a flour sample. In other embodiments, thesample is a stock sample. In some embodiments the sample is an amylaseconcentrate sample.

In some embodiments, the surface is selected from the group consistingof a tube, a centrifuge tube, a cuvette, a dipstick, a multiwell plate,a slide, a coverslip, a card, a bead, and a plate. In some embodiments,determining the level of hydrolysis of the detectably labeled starchsubstrate includes quantifying the soluble detectably labeled starchsubstrate fragments. In some embodiments, the method also includescalculating the α-amylase activity in the sample by correlating thequantity of soluble detectably labeled starch fragments to an α-amylasestandard. In some embodiments, the α-amylase standard is an α-amylasestandard curve.

In some embodiments, determining the level of hydrolysis of thedetectably labeled starch substrate includes quantifying the detectablylabeled starch substrate after separating the soluble detectably labeledstarch fragments from the reaction mixture. In some embodiments, themethod also includes releasing the detectably labeled starch substratefrom the surface after separating the soluble detectably labeled starchfragments from the reaction mixture. In certain embodiments, determiningthe level of hydrolysis of the detectably labeled starch substrateincludes quantifying the detectably labeled starch substrate afterreleasing the detectably labeled starch substrate from the surface. Insome embodiments, the method also includes calculating the α-amylaseactivity in the sample by correlating the quantity of detectably labeledstarch to an α-amylase standard. In some embodiments, the detectablylabeled starch substrate is a potato starch.

In some embodiments, the detectably labeled starch substrate includesD-glucose residues and is labeled on about one of every 300-1300D-glucose residues of the starch substrate. In some embodiments, thestarch substrate is detectably labeled with a label compound selectedfrom the group consisting of fluorescent, enzyme, radioactive, metallic,biotin, chemiluminescent, and bioluminescent molecules. In someembodiments, the label is a fluorophore. In some embodiments, thefluorophore is selected from the group consisting of5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS), FITC, andMarina Blue.

In some embodiments, the step of separating the soluble detectablylabeled starch fragments from the reaction mixture includes filteringthe reaction mixture to remove from the mixture detectably labeledstarch substrate. In certain embodiments, the step of filtering includesthe addition of a filtration aid selected from the group consisting ofresin, glass beads, beads, and celite. In some embodiments, the step ofseparating the soluble detectably labeled starch fragments from thereaction mixture includes centrifuging the reaction mixture to removefrom the mixture detectably labeled starch substrate. In someembodiments, the method also includes measuring an aliquot of thesupernatant of the centrifuged reaction mixture. In some embodiments,the step of separating the soluble detectably labeled starch fragmentsfrom the reaction mixture includes obtaining an aliquot of the reactionmixture and centrifuging the aliquot of the reaction mixture to removefrom the aliquot detectably labeled starch substrate. In certainembodiments, the step of separating the soluble detectably labeledstarch fragments from the reaction mixture includes contacting thefragments with an agent that binds to the detectably labeled starchfragments. In some embodiments, the agent is a lectin. In someembodiments, the agent is an antibody.

In some embodiments, the sample is an aqueous slurry. In someembodiments, the sample is contacted with the detectably labeled starchsubstrate for a reaction time of at least about 1 sec, 5 sec, 10 sec, 15sec, 20 sec, 25 sec, 30 sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 1min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11min, 12 min, 13 min, 14 min, 15 min, 16 min,17 min, 18 min, 19 min, 20min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29min, 30 min, 31 min, 32 min, 33 min, 34 min,35 min, 36 min,37 min, 38min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56min, 57 min, 58 min, 59 min, or 60 min. Preferably, the sample iscontacted with the detectably labeled starch substrate for a reactiontime of at least about 1 minute, at least about 5 minutes, at leastabout 10 minutes, or at least about 15 minutes.

According to yet another aspect of the invention, kits for measuringα-amylase activity in a sample are provided. The kits include a firstcontainer containing a detectably labeled starch substrate, a secondcontainer containing an α-amylase standard, instructions for measuringthe α-amylase activity in a sample.

According to another aspect of the invention, kits for measuringα-amylase in a sample are provided. The kits include a first containercontaining a detectably labeled starch substrate, calibration standards,and a conversion table or curve for converting fluorometer readings toamylase units.

In some embodiments of the foregoing kits, the sample is a flour sample.In other embodiments of the foregoing kits, the sample is a stocksample. In some embodiments of the foregoing kits the sample is anamylase concentrate sample. In some embodiments of the foregoing kits,the starch substrate is potato starch. In some embodiments of theforegoing kits, the detectably labeled starch substrate includesD-glucose residues and is labeled on about one of every 300-1300D-glucose residues of the starch substrate. In some embodiments of theforegoing kits, the detectable label is a label compound selected fromthe group consisting of fluorescent, enzyme, radioactive, metallic,biotin, chemiluminescent, and bioluminescent molecules. In certainembodiments of the foregoing kits, the starch substrate is detectablylabeled with a label compound selected from the group consisting offluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent,and bioluminescent molecules. In some embodiments of the foregoing kits,the label is a fluorophore. In certain embodiments of the foregoingkits, the fluorophore is selected from the group consisting of5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS), FITC, andMarina Blue. In some embodiments of the foregoing kits, the instructionsfor measuring the α-amylase activity in a sample recite a methodcomprising forming a reaction mixture by contacting a sample with adetectably labeled starch substrate for a time sufficient for α-amylasein the sample to hydrolyze the starch substrate, thereby releasingsoluble detectably labeled starch fragments, separating the solubledetectably labeled starch fragments from the reaction mixture, andquantifying the soluble detectably labeled starch as a measurement ofα-amylase activity in the sample. In some embodiments of the foregoingkits, the instructions further recite calculating the α-amylase activityin the sample by correlating the quantity of soluble detectably labeledstarch fragments to an α-amylase standard. In some embodiments, theα-amylase standard is an α-amylase standard curve.

In some embodiments of the foregoing kits, the step of separating thesoluble detectably labeled starch fragments from the reaction mixtureincludes filtering the reaction mixture to remove from the mixturedetectably labeled starch substrate. In some embodiments of theforegoing kits, the step of filtering includes the addition of afiltration aid selected from the group consisting of resin, glass beads,beads, and celite. In some embodiments of the foregoing kits, the stepof separating the soluble detectably labeled starch fragments from thereaction mixture includes centrifuging the reaction mixture to removefrom the mixture detectably labeled starch substrate. In someembodiments of the foregoing kits, the step also includes measuring analiquot of the supernatant of the centrifuged reaction mixture. Incertain embodiments of the foregoing kits, the step of separating thesoluble detectably labeled starch fragments from the reaction mixtureincludes obtaining an aliquot of the reaction mixture and centrifugingthe aliquot of the reaction mixture to remove from the aliquotdetectably labeled starch substrate. In some embodiments of theforegoing kits, the step of separating the soluble detectably labeledstarch fragments from the reaction mixture includes contacting thefragments with an agent that binds to the detectably labeled starchfragments. In some embodiments of the foregoing kits, the agent is alectin. In other embodiments of the foregoing kits, the agent is anantibody.

In some embodiments of the foregoing kits, the instructions furtherrecite that the sample is an aqueous slurry. In some embodiments of theforegoing kits, the instructions further recite that the sample iscontacted with the detectably labeled starch substrate for a reactiontime of at least about 1 sec, 5 sec, 10 sec, 15 sec, 20 sec, 25 sec, 30sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 1 min, 2 min, 3 min, 4 min,5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59min, or 60 min. Preferably, in some embodiments of the foregoing kits,the instructions recite that the sample is contacted with the detectablylabeled starch substrate for a reaction time at least about 1 minute, atleast about 5 minutes, at least about 10 minutes, or at least about 15minutes.

According to yet another aspect of the invention, methods of determiningamylase in a sample are provided. The methods include placing about 6 mlincubation buffer in a substrate tube, warming the substrate tube to 45°C., adding about 200 mg of the sample to the warmed substrate tube,incubating the sample mixture in the substrate tube 10 min at 45° C.,adding about 4 ml stop buffer to the sample mixture in the substratetube, filtering the stopped sample mixture into a container, determiningthe fluorescence in the filtrate, and optionally converting thefluorescence value into a Falling Number Equivalent value. In someembodiments, the container is a cuvette. In certain embodiments, thefluorescence is determined in a fluorometer. In some embodiments, theamylase comprises one or more amylases selected from the groupconsisting of cereal amylase, bacterial amylase, and fungal amylase. Insome embodiments, the sample selected from the group consisting of aflour sample, a stock sample, and an amylase concentrate sample. Incertain embodiments, the filtering is filtering through a microfiberfilter.

According to another aspect of the invention, methods of determiningamylase in a sample are provided. The methods include placing an about 3g sample into a first container, adding a sufficient amount of fungalincubation buffer to have the total weight of sample plus buffer equalof about 30 g, mixing the solution, extracting the solution for 5minutes at 45° C., adding the about 8 ml of the extract to a substratetube, incubating extract in substrate tube 10 minutes at 45° C., addingabout 2 ml stop buffer the tube, mixing the contents of the tube,filtering the mixture into a second container, determining thefluorescence in the filtrate, and optionally converting the fluorescencevalue into an Enzyme Units Equivalent value. In some embodiments, thefirst container is a tube. In certain embodiments, the second containeris a cuvette. In some embodiments, the fluorescence is determined in afluorometer. In some embodiments, the sample comprises one or moreamylases selected from the group consisting of cereal amylase, bacterialamylase, and fungal amylase. In some embodiments, the sample is selectedfrom the group consisting of a flour sample, a stock sample, and anamylase concentrate sample. In some embodiments, the flour sample is awheat flour sample. In some embodiments, the filtering is filteringthrough a microfiber filter.

According to yet another aspect of the invention, methods of determiningamylase in a sample are provided. The methods include placing about 200mg of the sample into a container, adding about 20 ml fungal incubationbuffer to the sample, mixing the sample solution, diluting about 2 ml ofthe solution with 10 ml incubation buffer in a container, optionallyfurther diluting the diluted solution to obtain a concentration withinrange of about 0.1-1.0 SKB unit/ml, placing about 8 ml of the dilutedsample into a container, incubating the about 8 ml diluted sample 10minutes at 45° C., adding about 2 ml stop buffer to the 8 ml dilutedsample, filtering the mixture through a filter into a detectioncontainer, determining the fluorescence in the filtrate, and optionallyconverting the fluorescence value into an Enzyme Units Equivalent valueand multiplying by the dilution factor as a measure of the originalamylase concentration. In certain embodiments, the container is a tube.In some embodiments, the detection container is a cuvette. In someembodiments, the fluorescence is determined in a fluorometer. In certainembodiments, the sample comprises one or more amylases selected from thegroup consisting of cereal amylase, bacterial amylase and fungalamylase. In some embodiments, the sample is selected from the groupconsisting of a flour sample, a stock sample, and an amylase concentratesample. In some embodiments, the flour sample is a wheat flour sample.In some embodiments, the filtering is filtering through a microfiberfilter.

According to another aspect of the invention, kits are provided. Thekits include a container containing a detectably labeled starchsubstrate, a standard, and/or a standard curve and/or a conversion tablefor converting fluorometer readings to amylase units, and instructionsfor using any of the aforementioned methods to determine the amount ofα-amylase activity in a sample. In some embodiments, the kits may alsoinclude buffers, tubes, calibration solutions, filters, and/or controlsamples. In some embodiments, the sample is selected from the groupconsisting of a flour sample, a stock sample, and an amylase concentratesample.

These and other aspects of the invention, as well as various embodimentsthereof, will become more apparent in reference to the drawings anddetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of results of amylase assay of flour samples atvarious concentrations versus the Falling Number of the samples.

FIG. 2 is a graph of the results of a determination of Doh Tone™concentration in wheat flours with the amylase assay.

FIG. 3 shows a graph of the results of a determination of pure Doh Tone™II (Code 416) with the amylase assay versus concentration.

FIG. 4 is a graph of the results of determination of Doh Tone™ (Code416) versus concentration to fit a quadratic curve.

FIG. 5 is a graph of the results of determination of Doh Tone™ (Code416) versus concentration to fit to a linear relation.

FIG. 6 is a graph illustrating the effect of using supernatants of aflour extract instead of flour suspensions as with the amylase assay.

FIG. 7 is a graph illustrating the effect of a stability test performedon AMYLease™ substrate at elevated temperature.

FIG. 8 is a table (FIG. 8A)and graph (FIG. 8B) illustrating the resultsof determination of fungal amylase from Sigma-Aldrich (St. Louis, Mo.).

FIG. 9 shows graphs of (FIG. 9A) results of the amylase assay on Sigmafungal amylase at high range of amylase units and (FIG. 9B) results ofthe amylase assay on Sigma fungal amylase at low range of amylase units.

FIG. 10 is a graph illustrating the effects of cereal amylase ondetermination of fungal amylase with the amylase assay.

DETAILED DESCRIPTION OF THE INVENTION

For the commercial and home use of flour for baking and food production,it is important to maintain an appropriate level of α-amylase activityin the flour. A level of activity that is too high may result in aproduct that is sticky and/or doughy and unmarketable; but flour withinsufficient α-amylase activity may not contain enough sugar for properyeast function, resulting in dry, crumbly bread. To augment the level ofendogenous α-amylase activity in flour, exogenous (e.g. substitute)α-amylase may be added to flour in the form of fungal α-amylase or otherα-amylase. Therefore, the ability to determine the level of activity ofboth endogenous (natural) and fungal α-amylase, or other α-amylase, in aflour sample would benefit the food production process and promote moreefficient use of flour in food production.

In addition to the use of grains and other plant products in baking,grains such as barley, oats, wheat, as well as plant components such ascorn, hops, and rice are used for brewing, both in industry and for homebrewing. The components used in brewing may be unmalted or may bemalted, which means partially germinated resulting in an increase in thelevels of enzymes including α-amylase. For successful brewing, adequatelevels of α-amylase enzyme activity are necessary to ensure theappropriate levels of sugars for fermentation.

As used herein, the term “flour” means milled or ground cereal grain.The term “flour” may also mean Sago or tuber products that have beenground or mashed. In some embodiments, flour may also contain componentsin addition to the milled or mashed cereal or plant matter. An exampleof an additional component, although not intended to be limiting, is aleavening agent. Cereal grains include: wheat, oat, rye, and barley, Inpreferred embodiments of the invention, the cereal grain is wheat. Tuberproducts include tapioca flour, cassava flour, and custard powder. Theterm “flour” also includes ground corn flour, maize-meal, rice flour,whole-meal flour, self-rising flour, tapioca flour, cassava flour,ground rice, and custard powder.

As used herein, the term “stock” means grains and plant components thatare crushed or broken. For example, barley used in beer production is agrain that has been coarsely ground or crushed to yield a consistencyappropriate for producing a mash for fermentation. As used herein, theterm “stock” includes any of the aforementioned types of plants andgrains in crushed or coarsely ground forms. It will be understood thatthe methods of the invention may be used to determine α-amylase activitylevels in flours, and also in stock, which includes the aforementionedtypes of grains, tubers, and other plant products that have beencrushed.

As used herein, the term “amylase concentrate” means a sample thatincludes amylase. The amylase may be fungal, bacterial, and/or cerealamylase. It will be understood that the methods of the invention may beused to determine the amount or level of α-amylase activity in a samplethat contains a single amylase and/or a sample that contains more thanone type of amylase, for example, the sample may include fungalα-amylase or may include cereal and fungal α-amylase.

The invention involves in some aspects, methods for measuring α-amylaseactivity in flour, grain or tuber products, stock, and amylaseconcentrate samples. As used herein, the term “α-amylase” meansendogenous α-amylase, exogenous α-amylase (e.g. α-amylase concentrate),or α-amylase that has been added to flour or stock. As used herein, theterm “α-amylase” means a protein having α-amylase activity, preferablyplant-derived α-amylase and/or microbial α-amylase. Plant-derivedα-amylase includes, but is not limited to, cereal α-amylase and wheatα-amylase. Microbial α-amylase includes, but is not limited to,bacterial α- amylase, and fungal α-amylase. As used herein, the term“α-amylase activity” means the enzymatic action of the α-amylase. Theenzymatic action of the α-amylase includes the hydrolysis (breakage) ofthe α-1,4, glucosidic bonds present in starch, which reduces the size ofthe starch molecules and converts the starch into sugar.

The invention involves in some aspects, contacting a sample with astarch substrate and determining the activity of the α-amylase enzyme ofthe sample in the breakdown of the starch substrate. As used herein, theterm “substrate tube” means a tube that contains a labeled starchsubstrate. In some embodiments, the sample is a flour sample. In someembodiments, the sample is a stock sample. In some embodiments, thesample is an amylase concentrate sample. In some aspects of theinvention, the starch substrate is detectably labeled. This detectablelabel is attached to the starch substrate utilizing standard chemistrymethods and allows quantification of the amount of cleavage of thestarch substrate by α-amylase after it is contacted with the sample.Such standard methods may include, but are not limited to, attaching adetectable label to the starch substrate through chemical conjugation.Various conjugation reagents including, but not limited to, cyanogenbromide activation or pryidinium dichromate oxidation, followed byreductive amination. In the case of cyanogen bromide activation, theactivated starch will react with the amino groups of fluorescentmaterials to form the fluorescene-labeled substrate starch.

In some embodiments, the activity of the α-amylase is determined byquantifying the amount of detectably labeled starch substrate fragmentsthat have been cleaved from the starch substrate. In other embodiments,the activity of the α-amylase is determined by quantifying the amount ofdetectably labeled starch substrate that remains intact followingcontact with the sample.

The invention involves in some aspects separating the soluble detectablylabeled starch fragments from the detectably labeled starch in thereaction mixture. Methods that may be used to separate the fragmentsfrom the reaction mixture include, but are not limited to: filtration,centrifugation, and affinity binding methods. As used herein, the term“filtering” means passing the sample through one or more filter devices.Such devices include, but are not limited to paper filters, screens,mesh, etc. Filtering may involve passing the material to be filteredthough a single filter, or through a multiple filters, which may be ofthe same type or may be of differing types (e.g., a screen followed by apaper or a mesh followed by a screen and/or paper filter). Filtration isdone using standard methods known in the art. One of ordinary skill inthe art will recognize there are numerous filtration methods,combinations, and techniques that are useful in the methods and kits ofthe invention. In the methods and kits of the invention, filteringmethods may also include the use of filtration aids including, but notlimited to: resins, beads including glass beads, and celite, which isalso known as diatomaceous earth and Kieselguhr. Selection and use ofsuch filtration aids in the methods of the invention, will be understoodby one of ordinary skill in the art.

The invention relates in part to the use of centrifugation methods toseparate detectably labeled fragments from the reaction mixture. Suchmethods include the centrifugation and may also include the removal ofan aliquot of the supernatant of the centrifugation for measurement ofthe amount of detectably labeled starch substrate fragments. The removalof an aliquot from the centrifuged reaction mixture may be followed bythe centrifugation of the aliquot prior to determination of the level ofdetectably labeled starch substrate in the aliquot.

The invention also relates in part to the use of affinity bindingmethods to separate detectably labeled fragments from the reactionmixture. An example of an affinity binding method, although not intendedto be limiting, is the use of affinity chromatography methods toseparate detectably labeled fragments from the reaction mixture.Affinity binding methods include the use of agents that bind to themolecules to be separated. Such agents include, but are not limited to,lectins and antibodies. As will be recognized by one of ordinary skillin the art, the agent may be bound to a support, e.g. as in affinitycolumn chromatography. It will be understood that in alternativeembodiments, the agent is not bound to a surface. Methods of separatingmolecules using methods such as affinity binding and/or affinitychromatography are well understood by those of ordinary skill in theart. Examples of affinity separation methods are provided in U.S. Pat.No. 6,362,008, which is hereby incorporated by reference in itsentirety.

One of ordinary skill in the art will recognize that following aseparation step as described herein, either the soluble detectablylabeled substrate fragments, the detectably labeled substrate, or both,can be measured using the methods of the invention to determine theα-amylase activity in the sample tested. Such measurements may be doneusing standard methods, including, but not limited to, transferring thesupernatant or filtrate samples to a measurement cuvette, followed bymeasurement on a calibrated fluorometer. In such readings, thefluorescent reading would be proportional to the amount of amylasepresented in the flour, stock, or amylase concentrate samples.

As used herein the term “time sufficient for α-amylase to hydrolyze thestarch substrate” means the amount of time for hydrolysis to occur. Thetime sufficient is at least about 1 sec, 5 sec, 10 sec, 15 sec, 20 sec,25 sec, 30 sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 1 min, 2 min, 3min, 4 min, 5 min, 6 min, 7 rain, 8 min, 9 min, 10 min, 11 min, 12 min,13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min,22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min,31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min,40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min,49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min,58 min, 59 min, or 60 min. Preferably, the time is at least 1 minute, atleast 5 minutes, at least 10 minutes, or at least 15 minutes.

As used herein the term: “hydrolysis” means at least partial hydrolysisof the starch substrate. Total hydrolysis of the starch substrate is notrequired. As used herein, the term “soluble detectably labeled starchfragments” means fragments of the detectably labeled starch that havebeen released from the starch by hydrolysis. The soluble detectablylabeled starch fragments are no longer attached to the starch substrate.

In some embodiments of the invention, a control sample, or amylasestandard may be prepared. As used herein the terms “amylase standard”and “control sample” means a sample with a known amount of α-amylaseactivity that may be contacted with a starch substrate identical to thatcontacted with the flour or stock test sample. The reaction with theknown amount of α-amylase activity thereby serves as a control reaction(or standard reaction) from which one of ordinary skill can extrapolatethe level of activity in the test sample. One of ordinary skill in theart will recognize how to prepare and utilize a control or standardreaction to allow determination of the α-amylase activity in testsamples.

The invention also includes in some aspects, the use of an α-amylasestandard curve, which may include, for example, fluorescent values thatcorrespond to a range of α-amylase concentrations. An α-amylase standardcurve may be used to compare the value in a sample as a determination ofthe amount of α-amylase activity in the sample. In some embodiments, a“control” or “amylase standard” value is a value from an α-amylasestandard curve.

The invention includes a starch substrate that is detectably labeled. Asused herein, a “starch substrate” is a starch molecule upon whichα-amylase acts enzymatically. As used herein, the term “starch”includes, but is not limited to, wheat starch, waxy wheat starch, cornstarch, waxy maize starch, oat starch, rice starch, tapioca starch,mung-bean starch, potato or high amylose starches, and sorghum starch.In preferred embodiments, the starch substrate is potato starch.. Insome preferred embodiments, the potato starch is cross-linked potatostarch.

As used herein, a starch substrate or starch substrate fragment that is“detectably labeled” means a starch substrate or substrate fragment towhich a label that can be detected is attached. The term “label” as usedhere means a molecule preferably selected from, but not limited to, thegroup consisting of fluorescent, enzyme, radioactive, metallic, biotin,chemiluminescent, and bioluminescent molecules. As used herein, thelabel is not a colorimetric label, e.g., a chromophore molecule. In someaspects of the invention, a label may be a combination of the foregoingmolecule types.

Radioactive or isotopic labels include, for example, ¹⁴C, ³H, ³⁵S, ¹²⁵I,and ³²P. Fluorescent labels include any compound that emits anelectromagnetic radiation, preferably visible light, resulting from theabsorption of incident radiation and persisting as long as thestimulating radiation is continued. Such compounds include coumarincontaining molecules, and further include anthroyl compounds,naphthalene compounds, pyrene compounds, compounds containing benzyl,pyrenyl and phenyl groups, fluorescein compounds, anthracene compounds,compounds containing conjugated pi electron systems, but are not limitedto these categories of compounds and include any compound that could beused as a label in this invention.

Examples of the fluorescent coumarin molecules include7-hydroxycoumarin, 7-aminocoumarin, and further include6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid, succinimidylester,7-amino-3-((((succinimidyl)oxy)carbonyl)methyl)-4-methylcoumarin-6-sulfonicacid, 7-diethylaminocoumarin-3-carboxylic acid,7-diethylaminocoumarin-3-carboxylic acid succinimidyl ester,7-diethylamino-3-(4′-isothiocyanophenyl)-4-methylcoumarin,7-dimethylaminocoumarin-4-acetic acid, 7-dimethylaminocoumarin-4-aceticacid succinimidyl ester, 7-hydroxycoumarin-3-carboxylic acid,7-hydroxycoumarin-3-carboxylic acid succinimidyl ester,7-hydroxy-4-methylcoumarin-3-acetic acid,7-hydroxy-4-methylcoumarin-3-acetic acid succinimidyl ester,7-methoxycoumarin-3-carboxylic acid, 7-methoxycoumarin-3-carboxylic acidsuccinimidyl ester, 7-diethylaminocoumarin-3-carbonyl azide and7-methoxycoumarin-3-carbonyl azide.

Examples of naphthalene compounds include5((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS),6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid,2-dimethylaminonaphthalene-5-sulfonyl chloride,dimethylaminonaphthalene-6-sulfonyl chloride,6-(N-methylanilino)naphthalene-2-sulfonyl chloride,6-(p-toluidinyl)naphthalene-2-sulfonyl chloride and 5-acenaphthalene.

Examples of other fluorescent labels include but not limited to2,4-dinitrophenyl, acridine, cascade blue, rhodamine, 4-benzoylphenyl,7-nitrobenz-2-oxa-1,3-diazole,4,4-difluoro-4-bora-3a,4a-diaza-3-indacene and fluorescanine.Absorbance-based labels include molecules that are detectable by thelevel of absorption of various electromagnetic radiation. Such moleculesinclude, for example, the fluorescent labels indicated above.

Chemiluminescent labels in this invention refer to compounds that emitlight as a result of a non-enzymatic chemical reaction.

As used herein, fluorophores include, but are not limited toamine-reactive fluorophores that cover the entire visible andnear-infrared spectrum. Examples of such fluorophores include, but arenot limited to, 4-methylumbelliferyl phosphate, fluoresceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),BODIPY dyes; Oregon Green, rhodamine green dyes; the red-fluorescentRhodamine Red-X, Texas Red dyes; and the UV light-excitable CascadeBlue, Cascade Yellow, Marina Blue, Pacific Blue and AMCA-X fluorophores.Fluorophores may also include non-fluorescent dyes used in fluorescenceresonance energy transfer (FRET).

In addition to alkaline phosphatase and peroxidase, other enzymes thatcan be used in methods and kits of the invention include, but are notlimited to β-galactosidase, β-glucuronidase, β-glucosidase,β-glucosidase, α-mannosidase, galactose oxidase, glucose oxidase andhexokinase.

The labeled molecules of the invention can be prepared from standardmoieties known in the art. As is recognized by one of ordinary skill inthe art, the labeling process will vary according to the molecularstructure of the detectable label. For fluorescent materials with freeamino groups, a typical process, though not intended to be limiting,would be to use alkaline cyanogen bromide to activate starch substrate.The cyanogen-bromide-activated starch reacts with the free amino groupof the fluorophores to form a new bond, e.g. an isourea, carbamate, orimidocarbamate bond, which links the fluorophores onto the starchsubstrate. Other methods of labeling molecules with one or more of theabove-identified types of detectable labels are routinely used and arewell understood by those of ordinary skill in the art.

The invention involves, in some embodiments, a labeled starch substratethat is labeled on about one of every 300-1300 D-glucose residues of thestarch. In certain embodiments, the starch substrate is labeled on aboutone of every 300-500, about one of every 500-700, about one of every700-900, about one of every 900-1100, about one of every 1100-1300, orvarious combinations thereof. Smaller ranges also are contemplated, suchas every 100 units (300-400, 400-500, etc.), every 50 units (300-350,350-400, etc.), and so on.

The invention in another embodiment, includes measuring α-amylaseactivity in a flour, stock, or amylase concentrate sample by forming areaction mixture by contacting the sample with a detectably labeledstarch substrate attached to a surface, for a time sufficient for theα-amylase enzyme to hydrolyze the starch substrate. As used herein theterm “surface” means a material including any synthetic or naturalmaterial. Examples of surfaces of the invention include, but are notlimited to: glass, plastic, nylon, metal, paper, cardboard, and can bein numerous forms including, but not limited to, tubes, centrifugetubes, cuvettes, cards, slides, dipsticks, beads, coverslips, multiwellplates, Petri plates, etc. One of ordinary skill in the art willrecognize that numerous additional types of surfaces can be used in themethods of the invention.

As used herein the term “attached to a surface” means chemically orbiologically linked to the surface and not freely removable from asurface. Examples of attachment, though not intended to be limiting arecovalent binding between the surface and the starch substrate,attachment via specific biological binding, or the like. For example,“attached” in this context includes chemical linkages,chemical/biological linkages, etc. As used herein the term “covalentlyattached” means attached via one or more covalent bonds. As used hereinthe term “specifically attached” means a species is chemically orbiochemically linked to a surface as described above with respect to thedefinition of “attached,” but excluding all non-specific binding. In themethods of the invention, a starch substrate that is attached to asurface is attached such that the substrate is not removable from thesurface without specific stripping methods or solutions. Such strippingmethods may include, but are not limited to, physical methods such asscraping or heating, enzymatic methods, and chemical methods, which mayinclude but are not limited to contacting the attached substrate andsurface with a solution such that the link between the substrate and thesurface is broken and the substrate is released.

One of ordinary skill in the art will be able to envision the steps offorming a reaction mixture by contacting a detectably labeled starchsubstrate attached to a surface with an α-amylase enzyme and removingthe labeled fragments from the reaction mixture. The amount of labelpresent on the fragments released by the hydrolysis (soluble fragments)is measured and/or the amount of label that remains on the starch thathas not been hydrolyzed and therefore remains attached to the surface ismeasured, and either or both measurements are to be compared to theinitial amount of label on the surface prior to contact with theα-amylase enzyme. From a comparison of the levels of labeled starchbefore and after hydrolysis, a determination of the amount of α-amylaseactivity in the reaction mixture can be made. One of ordinary skill inthe art will recognize that the total amount of detectably labeledstarch prior to contact with the α-amylase, can be compared with eitherthe level of label on pieces released by α-amylase hydrolysis, or theamount of detectably labeled substrate that remains attached to thesurface following hydrolysis. This type of method can be used todetermine the amount of α-amylase enzyme activity in the flour or stocksample or control sample.

The following illustrates the use of a method of the invention todetermine the level of α-amylase activity in a flour, stock, and/oramylase concentrate sample. For example, if detectably labeled starch iscontacted with a flour, stock and/or amylase concentrate sample for atime sufficient to hydrolyze the starch and subsequent measurement ofthe amount of detectably labeled starch substrate fragments that are notattached to the surface is determined to be zero, it indicates theabsence of α-amylase activity in the flour, stock, and/or amylaseconcentrate sample tested. In addition, the determination that theoriginal amount of detectably labeled starch substrate that was attachedto the surface remains attached to the surface, indicates that there isno α-amylase activity in the sample. In contrast, if the sample containsα-amylase, the enzyme will break down the starch substrate and thehydrolyzed substrate fragments will be released or solubilized. Afterseparation of the hydrolyzed, small-sized starch fragments from thenon-hydrolyzed starch substrate, fluorescence from either the starchfragments or the non-hydrolyzed starch substrate can be measured todetermine quantity of α-amylase activity in the sample. The amount ofα-amylase activity in the flour, stock, and/or amylase concentratesample will be positively proportional to the fluorescent reading in thestarch fragments, but inversely proportional to the fluorescence in thenon-hydrolyzed starch substrate.

In some embodiments of the invention, the reaction mixture includesdetectably labeled starch substrate attached to a surface such as a testtube or centrifuge tube, which is contacted with α-amylase in a sample.Following contact for a time sufficient for α-amylase to hydrolyze thestarch substrate, the hydrolyzed starch substrate fragments that are notattached to the surface can be separated from the non-hydrolzyedsubstrate and measured, and/or the detectably labeled starch substratethat remains attached to the surface may be measured as attached to thetube, or may be stripped off the surface and its quantity determined.For example, to strip off the detectably labeled starch substrateattached to the surface, the surface may be treated with physical orchemical methods. The amount of stripped detectably labeled starchsubstrate is then collected and the level of labeled starch substrate ismeasured as a determination of the activity level of α-amylase in thesample. One of ordinary skill in the art will recognize that prior todetermination the activity level, a purification step such as, but notlimited to, centrifugation, filtration, or affinity binding methods, maybe used to further separate the soluble detectably labeled fragments.Following the separation, a determination of the amount of solubledetectably labeled fragments and/or retained substrate is done. Thisdetermination may be done using a routine detection method, which can beselected based on the type of detectable label utilized. Examples ofsuch methods, include, but are not limited to the use of a fluorometerto determine the amount of detectably labeled fragments or retainedsubstrate when the label is fluorescence, or the use of a scintillationcounter if the label is radioactive. One of ordinary skill in the artwill be familiar with the variety of detection systems that can beutilized in the methods of the invention.

The invention also relates in some aspects to kits for measuringα-amylase activity in a flour. stock, and/or amylase concentrate sample.The kits of the invention may include a first container of detectablylabeled starch substrate, a second container of an α-amylase standardand instructions for measuring the α-amylase activity in a flour, stock,and/or amylase concentrate sample. Some kits of the invention mayinclude a container containing a starch substrate, a second containercontaining an α-amylase standard, a third container containing adetectable label, instructions for labeling the starch substrate, andinstructions for measuring the α-amylase activity in a flour, stock,and/or amylase concentrate sample. The kits of the invention may alsoinclude additional components such as tubes, vials, containers, dipsticks, buffers, water, fluorometer calibration standards, an α-amylasestandard curve, etc. The kits of the invention may also be provided inconjunction with supplementary equipment (e.g. measuring devices such asfluorometers), and may also include instructions for running the assaysof the invention utilizing the supplementary equipment.

EXAMPLES Example 1 Introduction

Wheat or fungal α-amylase activity in flour samples is tested. Themethod may also be used to measure the activity of other types ofmicrobial α-amylase, such as bacterial α-amylase activity.

Methods

Preparation of a Fluorescent Starch Substrate for Use in Assayingα-amylases

Starch, for example potato starch or waxy maize starch, is activated byreaction with alkaline cyanogen bromide using the method of Cuatrecasas,P. and Anfinsen, C., Meth. Enzymol. 22:351-378, 1971. Activation isfollowed by reaction with a fluorescent dye that has a free amino group.The amino group of the fluorescent molecule reacts with thecyanogen-bromide-activated starch according to the following reactions,wherein R=ligand (e.g. fluorescent or enzyme).

The amount of derivatization of the starch is kept to 1 in 300 to 1 in1300 D-glucose residues to allow α-amylase to react with the derivatizedstarch. The labeling ratio can be determined by NMR using standardmethods. It can also be determined by measuring the quantity offluorescence labeling of D-glucose residues. The number of fluorescentmolecules can be calculated by the absorbance coefficient, while thenumber of micromoles of glucose can be determined by the microphenol-sulfuric acid method using standard procedures.

Assay for α- amylase Activity (A)

Water is added to a flour sample to liquefy and make a slurry. A knownamount of prepared liquefied flour is added to an aliquot of preparedsubstrate (fluorescent starch). The mixture is incubated at 45° C. Thereaction mixture is added to a filtration device that will retain thestarch and flour particles but permit hydrolyzed detectably labeledfragments to pass. The filtrate is mixed well and read in a calibratedfluorometer to determine amylase activity.

Assay for α-amylase Activity (B)

-   -   1) 200 mg fluorescent starch is suspended in 1 ml buffer.    -   2) The 1-ml sample to be assayed is added to the starch.    -   3) The samples are mixed and reaction allowed to proceed.    -   4) The reaction mixture is then mixed again and centrifuged and        aliquots are taken from the supernatant for measurement of the        fluorescence. The level of fluorescence is proportional to the        amount of α-amylase activity in the sample.    -   5) Alternatively, samples are continuously stirred and aliquots        are taken as described in step 3, centrifuged, and fluorescence        measured as in step 4.

Example 2 Methods

The following buffers were used as indicated in the Examples below.

Reaction Buffer Stock was prepared by dissolving 17.6 ml Acetic acid,16.0 gm anhydrous sodium acetate, 29.2 gm sodium chloride, and 5.6 gmcalcium chloride in de-ionized (DI) water to a final volume of 1 liter.The Reaction Buffer used in the assays was prepared by diluting theReaction Buffer Stock 1:10 v/v with DI.

Stop Buffer was prepared by mixing 115.5 ml acetic acid with DI water toa final volume of 1 liter.

Phosphate Buffer (Reaction Buffer for Cereal Amylase) was prepared bydissolving 1.2651 gm anhydrous monobasic sodium phosphate and 1.562 gmanhydrous dibasic sodium phosphate in DI to a final volume of 1 liter.

Substrate Tubes used in the reactions were AMYLease™ substrate tubes(Vicam, Watertown, Mass.) containing labeled starch substrate.

Test for Cereal Amylase

Introduction

The objective of this study was to measure cereal amylase activity.

The fluorometer was calibrated and 6 ml Phosphate Buffer was added toeach AMYLease™ substrate tube (Vicam). The substrate tubes werepre-warmed to 45° C. 200 mg of sample was weighed and added to thepre-warmed substrate tube. The mixture was incubated exactly 10 minutesat 45° C. 4 ml Stop Buffer was added to the tube following theincubation.

The incubation mixture was filtered through a microfiber filter into aclean cuvette. The fluorescence was read in the calibrated fluorometerand the fluorescence resulting values, which are shown in Table 1 wereplotted versus the Falling Number values for a series of known samples(see FIG. 1). TABLE 1 Cereal Amylase test results. Falling NumberFluorescence (ppm) 570 33 524 31 354 53 280 63 272 54 132 170 118 220111 260

Example 3

Test For Fungal Amylase in Wheat Flour and Fungal Concentrates

Introduction

The objective of this study was to utilize to measure fungal amylaseactivity in wheat flour and in concentrated fungal amylase preparations(e.g., amylase concentrates). Tests were conducted with two types ofcommercial fungal amylase preparations. Doh Tone™ (American Ingredients,Inc. Anaheim, Calif.) contains 5.5% by weight neat fungal amylase, andit also contains fungal proteinases, wheat starch and silica. Doh Tone™is usually dosed at 2 g per 100 lb wheat flour. Doh Tone™ II (AmericanIngredients, Inc.) contains 2.75% by weight of neat fungal amylase,wheat starch and silica. Doh Tone™ II does not contain proteinases. DohTone™ II is usually dosed at 4 g per 100 lb wheat flour.

Example 3A

This study encompassed linearity, precision, and day-to-dayrepeatability for measurement of fungal amylase in wheat flour.Linearity was determined using results obtained from flour samplesspiked with Doh Tone™ II at levels from 0.002% to 0.15 % by weight.Precision was determined by making triplicate measurements on floursamples spiked at 0.008% by weight with Doh Tone™ II and samples spikedat 0.004% by weight with Doh Tone™. Day-to-day repeatability over 4 dayswas assessed by testing flour samples spiked with Doh Tone ™ II at0.008%, 0.02%, 0.04%, 0.08% and 0.15% by weight.

Methods

Fungal Amylase in Wheat Flour

1. A 1 mg/ml suspension of Doh Tone™ or Doh Tone™ II in Reaction Bufferwas prepared.

2. A 3 g (±0.05 g) flour sample to be tested was added to a 50-ml tube.The appropriate volume of Doh Tone™ suspension was added to give thedesired final % by weight in the 3 g flour sample. Reaction Buffer wasadded until the total weight of sample plus buffer was 30 g (±0.05 g).The mixture was mixed well by capping and inverting the tube severaltimes. The extract was then allowed to settle for 3-5 minutes.

3. 8 ml of the extract was placed in an empty tube and warmed to 45° C.in a heating block. The heating took about 3-5 minutes.

4. The pre-warmed extract was added to a Substrate Tube. The SubstrateTube was capped and rapidly mixed by inverting tube 4-6 times, andplaced in the heating block for a 10-min incubation.

5. After incubating exactly 10 minutes, 2 ml Stop Buffer was added tothe Substrate Tube. The Substrate Tube was capped and rapidly mixed byinverting tube 4-6 times.

6. For each sample, one microfiber filter (Vicam part # 31955, Vicam,Watertown, Mass.) was placed into a filter funnel (Vicam part # 36020).The incubation mixture was filtered through microfiber filters into aclean cuvette.

7. The fluorescence of samples was measured in a fluorometer calibratedusing calibration standards (Vicam part# 33060) and with the high (red)standard set to 1000 ppm and the low (green) standard set to 0 ppm.

Results

Linearity

Linearity was determined using spiked wheat flour samples ranging from0.002% to 0.15 % Doh Tone™ II by weight run as described above. Table 2illustrates the results at different Doh Tone™ percentages in flour.FIG. 2 is a graph that of the linear regression analysis equation forthe fluorescence reading versus the amount of Doh Tone™ II spiked. Thecorrelation coefficient (r) of 0.998 from the above linear regressionequation indicates that the linearity of this method is very good forDoh Tone™ II in flour in the range 0.002% to 0.15% by weight. TABLE 2Results of tests to determine linearity of assays Doh Tone ™ Percentagein Flour (Karl98) Fluorescence-ppm 0 110 0.002 120 0.004 130 0.008 1500.02 200 0.04 280 0.08 490 0.1 580 0.15 850Precision

Precision was determined using the method provided above. Triplicatemeasurements were made on flour samples spiked at 0.008% by weight withtwo different lots of Doh Tone™ II and on samples spiked at 0.004% byweight with two different lots of Doh Tone™. Table 3 shows the resultsof this study. The results above show a good precision for differentlots of commercial fungal amylase preparations in flour or neat inbuffer. TABLE 3 Results of tests to determine assay precision C.VSamples Lot# Repeat-1 Repeat-2 Repeat-3 Average STDEV (%) Flour K99 w/o95 90 89 91.33 3.21 3.52% Doh Tone ™ Flour K99 w/ 0.004% 2253 130 120120 123.33 5.77 4.68% Doh Tone ™ Flour K99 w/ 0.004% 3035 140 130 130133.33 5.77 4.33% Doh Tone ™ Flour K99 w/ 0.008% 2225 130 120 120 123.335.77 4.68% Doh Tone ™ II Flour K99 w/ 0.008% 3035 140 130 130 133.335.77 4.33% Doh Tone ™ IIw/o = without; w/ = with; STDEV = standard deviation; C.V. = coefficientof varianceRepeatability

Day-to-day repeatability over 4 days was assessed by testing floursamples spiked with Doh Tone 2 at 0.008%, 0.02%, 0.04%, 0.08% and 0.15%by weight using the method provided above. Table 4 shows the results ofthis study. The results indicate a good repeatability of measurements ofcommercial fungal amylase in flour samples. TABLE 4 Results of tests todetermine repeatability of assays. FL. FL. FL. FL. Day- Day- Day- Day-Aver- C.V. Samples 1 2 3 4 age STDEV (%) Flour K98 100 110 110 99 104.756.08 5.80% w/o Doh Tone ™ (DT) Flour K98 140 150 140 140 142.50 5.003.51% w/0.008% DT C416 Flour K98 190 200 190 190 192.50 5.00 2.60%w/0.02% DT C416 Flour K98 280 280 280 280 280.00 0.00 0.00% w/0.04% DTC416 Flour K98 490 490 500 500 495.00 5.77 1.17% w/0.08% DT C416 FlourK98 n/a 850 930 890 890.00 40.00 4.49% w/0.15% DT C416w/o = without; w/ = with; FL = Fluorescence; STDEV = Standard deviation;C.V. = coefficient of variance

Example 3B

This study determined linearity and precision of measurements ofconcentrated commercial preparations of fungal amylase. Linearity wasdetermined using results obtained from diluted suspensions of Doh Tone™II over a concentration range of 0.01 to 1.0 gm/ml. Precision wasassessed using triplicate measurements of 0.1 mg/ml solutions of DohTone™ I and Doh Tone™ II.

Concentrated Fungal Amylase Procedure

1. Dilution steps were required for commercial preparations that maycontain 2-5% by weight of neat fungal amylase.

2. The fluorometer was calibrated suing calibration standards (Vicampart# 33060) and with the high (red) standard set to 1000 ppm and thelow (green) standard set to 0 ppm.

3. 200 mg (±1 mg) of fungal amylase preparation to be tested was placedin a 50-ml tube. 20 ml Reaction Buffer was added to the tube. The tubewas capped and mixed well by inverting the tube several times. 2 ml ofthe this first dilution was added to another tube and diluted with 18 mlReaction Buffer.

4. 1.6 ml of the second dilution was placed in an empty tube, 6.4 mlFungal Incubation Buffer was added, and the tube was warmed to 45° C. inthe heating block. The warming took from 3-5 minutes.

[Note: In some experiments, a series of further dilutions of the seconddilution and Reaction Buffer were prepared to create a range ofconcentrations to obtain a concentration within range of about 0.1-1.0Sandstedt-Kneen-Blish (SKB) unit/ml for testing (see FIGS. 2-4). An SKBunit is an α-amylase dextrinizing unit (DU), and is defined as thequantity of α-amylase that will dextrinize soluble starch in thepresence of an excess of β-amylase at the rate of 1 gram per hour at 30°C. [see Institute of Medicine Food Chemicals Codex, 4th Ed., pp.1451-1454, National Academy Press, Chapman & Hall, CRC netBASE; and R.M. Sandstedt, et al., Cereal Chemistry 16:712-723 (1939)]. The stepsprovided below were followed for each of the dilutions to determine theamount of amylase at each dilution].

5. The prewarmed extract was added to a Substrate Tube, which was cappedand rapidly mixed by inverting tube 4-6 times. The Substrate Tube wasplaced in the heating block for a 10-minute incubation.

6. After incubating exactly 10 minutes, 2 ml of Stop Buffer was added.The Substrate Tube was capped and rapidly mixed by inverting tube 4-6times.

7. For each sample, one microfiber filter (Vicam part # 31955) wasplaced into a filter funnel (Vicam part # 36020). The incubation mixturewas filtered through microfiber filters into a clean cuvette.

8. The fluorescence of samples was measured in a fluorometer calibratedusing calibration standards (Vicam part# 33060) with the high (red)standard set to 1000 ppm and the low (green) standard set to 0 ppm.

9. In some instances, the fluorescence value of a sample is convertedinto an Enzyme Units Equivalent value. The conversion is done by readingoff a plot (e.g. standard curve) of fluorescence value versus fungalamylase concentrations standardized in SKB units, which is generatedusing a series of fungal amylase samples of known concentrations. Thestandard curve is used to determine the equivalent Enzyme Unit value fora test (unknown) sample, based on the fluorescence determined for thesample using the α-amylase assay.

The precision and linearity of measurement of concentrated fungalamylase preparations was determined using the procedure described above.

Linearity of Measurements of Concentrated Fungal Amylase

Linearity was determined using results obtained from diluted suspensionsof Doh Tone™ II over a concentration range of 0.01 to 1 mg/ml using theprocedure described above (See Table 5). FIG. 3 shows the determinationof Pure Doh Tone™ II with the assay. As expected for a substrate-basedenzyme assay there is a working range, which appears to be from 0.01 to0.1 mg/ml of Doh Tone™ II. FIG. 4 and FIG. 5 show the curves of thequadratic and linear relationship of fluorescence to Doh Tone™concentration in mg/ml), respectively. Within the working range, thecurve is better fit by a quadratic than by a linear relationship. TABLE5 Results of linearity tests Doh Tone ™ (DT) Conc. (mg/ml) Fluorescence(ppm) 0 14 0.01 17 0.02 22 0.04 31 0.05 38 0.08 60 0.1 78 0.15 150 0.2230 0.3 410 0.4 600 0.5 770 0.6 930Precision of Measurements of Concentrated Fungal Amylase

Triplicate measurements were made of Doh Tone™ II or Doh Tone™ atconcentrations of 0.1 mg/ml. Table 6 shows the results of this study.The results indicated a good precision for different lots of commercialfungal amylase preparations in flour or neat in buffer. TABLE 6 Resultsof tests to determine assay precision using fungal concentrate Re- Re-Re- peat- peat- peat- Aver- C.V Samples Lot# 1 2 3 age STDEV (%) 0.1mg/mL 3097 330 330 340 333.33 5.77 1.73% Pure Doh Tone ™ 0.1 mg/mL 2213100 110 100 103.33 5.77 5.59% Pure Doh Tone ™ IISTDEV = standard deviation; C.V. = coefficient of variance

Discussion Examples 3A and 3B

Linearity

The linearity using this method for fungal amylase in wheat flour isvery good as indicated by the correlation coefficient (r) of 0.998.

Precision

The results of the assays showed a very good precision for fungalamylase in flour with CVs less than 5% for Doh Tone™ II concentrationsranging from 0.008% to 0.15%

Repeatability

Repeatability for fungal amylase in flour was very good withcoefficients of variance (C.V.s) ranging from 0.0% to 4.49% fordifferent concentrations of amylase.

Linearity for Concentrated Fungal Preparation

The concentrated preparation showed an excellent fit to a quadraticrelationship R²=0.999 and a good fit (R²=0.98) to a linear relationship.

Example 4

Comparisons of different lots of Doh Tone™ and Doh Tone™ II (AmericanIngredients, Inc) were done using the methods described above. Resultsfrom various lots are shown in Table 6. TABLE 6 Results of assaycomparison of Doh Tone ™ and Doh Tone ™ II Doh Tone ™ (0.2 mg/ml)Fluorescence (ppm) DT LOT # 415303601 860 DT LOT # 415303501 830 DT LOT# 415309701 830 DT LOT # 415225301 630 DT-II LOT # 416221301 260 DT-IILOT # 416222501 260 DT-II LOT # 416303501 300 DT-II LOT # 416227401 230DT-II LOT # Code 416 260 DT-II LOT # 416022101 (o) 320 DT LOT #415022101 (o) 840DT = Doh Tone ™

Example 5

Tests were performed to compare results of the amylase assay performedon flour supernatant versus flour suspension. Various flour samples withdifferent percentages of Doh Tone™ and Doh Tone™ II were tested usingthe methods provided above (see Example 3). In that procedure the samplewas allowed to settle during the extraction procedure and an 8 ml sampleof the supernatant was taken for pre-warming. Duplicate samples weretested using a modification of the extraction method whereby the samplewas rapidly mixed by inverting the tube 4-6 times prior to talking an 8ml sample for pre-warming. The results are provided in Table 7 and agraph of the comparison of the results is shown in FIG. 6. The resultsindicate that there is a consistent proportional increase in thefluorescence if a whole suspension of extract is used as compared to asupernatant. TABLE 7 Results of sample supernatant versus samplesuspension assay. Samples Suspension Supernatant Difference Flour Karl99 130 100 30 0.0005% DT in K99 140 110 30 0.008% DT II in K99 160 13030 0.04% DT II in K99 270 230 40 0.08% DT II in K99 500 400 100 0.001%DT in K99 160 130 30 0.0005% DT in K99 140 110 30K99 = flour sample Karl 99; DT = Doh Tone ™

Example 6

A stability test was performed on an amylase substrate (Vicam, lot03PF15006) at elevated temperature using the assay methods providedabove. Flour samples (K98) were dosed with various amounts of fungalamylase (FA). The samples were then assayed as described in Example 3above using substrate stored normally or substrate that had been kept at37° C. for 4 days as an accelerated stability condition. The results areshown in FIG. 7.

Example 7

Tests were conducted using the methods described above to determine theworking range of the amylase assay on fungal amylase from Sigma (St.Louis, Mo.). Results are shown in FIGS. 8A and 8B, which show theresults in fluorescence per unit of the Sigma fungal amylase. FIGS. 9Aand 9B are graphs of the results of the amylase assay on Sigma fungalamylase at high range of amylase units (FIG. 9A) and a low range ofamylase units (FIG. 9B).

Example 8

Tests were run to determine the effects of native cereal amylaseactivity already in flour on the determination of fungal amylase usingthe amylase assay. Flours with different amounts of native cerealamylase activity (Falling Number from 250-524) were spiked with fungalamylase at various dosages and assayed using the amylase assay asdescribed in Example 3. Most of the signal was contributed by the nativecereal amylase. The signal from an untreated flour blank was subtractedfrom the total value to obtain a value for the added fungal amylase. Theresults are indicated in FIG. 10 and Table 8. TABLE 8 Results of amylaseassay of fungal amylase in flours containing different amounts of nativecereal amylase activity DT II Buffer FN 524 FN 354 FN 272 FN 250 Conc(%) Buffer FN 524 FN 354 FN 272 FN 250 (−B) (−B) (−B) (−B) (−B) 0 16 99315 520 625 0 0 0 0 0 0.004 18 120 325 525 615 2 20 10 5 0.008 21 140350 550 670 5 40 35 30 45 0.015 25.5 170 375 590 690 9.5 70 60 70 650.02 29 195 400 630 785 13 95 85 110 160 0.04 44 290 560 720 925 28 190245 200 300DT II = Doh Tone ™ II; FN = Falling Number value; (−B) = value minus theblank

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1. A method for measuring α-amylase activity in a sample, comprisingforming a reaction mixture by contacting a sample with a detectablylabeled starch substrate for a time sufficient for α-amylase in thesample to hydrolyze the starch substrate, thereby releasing solubledetectably labeled starch fragments, separating the soluble detectablylabeled starch fragments from the reaction mixture, and determining thelevel of hydrolysis of the detectably labeled starch substrate as ameasurement of α-amylase activity in the sample.
 2. The method of claim1, wherein the sample is selected from the group consisting of a floursample, a stock sample, and an amylase concentrate sample.
 3. The methodof claim 1, wherein determining the level of hydrolysis of thedetectably labeled starch substrate comprises quantifying the detectablylabeled starch substrate.
 4. The method of claim 1, wherein determiningthe level of hydrolysis of the detectably labeled starch substratecomprises quantifying the soluble detectably labeled starch substratefragments.
 5. The method of claim 1, further comprising calculating theα-amylase activity in the sample by correlating the quantity ofdetectably labeled starch to an α-amylase standard.
 6. The method ofclaim 1, further comprising calculating the α-amylase activity in thesample by correlating the quantity of soluble detectably labeled starchfragments to an α-amylase standard.
 7. The method of claim 1, whereinthe detectably labeled starch substrate is potato starch.
 8. The methodof claim 1, wherein the detectably labeled starch substrate comprisesd-glucose residues and is labeled on about one of every 300-1300d-glucose residues of the starch substrate.
 9. The method of claim 1,wherein the starch substrate is detectably labeled with a label compoundselected from the group consisting of fluorescent, enzyme, radioactive,metallic, biotin, chemiluminescent, and bioluminescent molecules. 10.The method of claim 9, wherein the label is a fluorophore.
 11. Themethod of claim 10, wherein the fluorophore is selected from the groupconsisting of 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid(EDANS), fluorescein isothiocyanate (FITC), and Marina Blue.
 12. Themethod of claim 1, wherein the step of separating the soluble detectablylabeled starch fragments from the reaction mixture comprises filteringthe reaction mixture to remove from the mixture detectably labeledstarch substrate.
 13. The method of claim 12, wherein the step offiltering includes the addition of a filtration aid selected from thegroup consisting of resin, glass beads, beads, and celite.
 14. Themethod of claim 1 , wherein the step of separating the solubledetectably labeled starch fragments from the reaction mixture comprisescentrifuging the reaction mixture to remove from the mixture detectablylabeled starch substrate.
 15. The method of claim 14, further comprisingmeasuring an aliquot of the supernatant of the centrifuged reactionmixture.
 16. The method of claim 1, wherein the step of separating thesoluble detectably labeled starch fragments from the reaction mixturecomprises obtaining an aliquot of the reaction mixture and centrifugingthe aliquot of the reaction mixture to remove from the aliquotdetectably labeled starch substrate.
 17. The method of claim 1, whereinthe step of separating the soluble detectably labeled starch fragmentsfrom the reaction mixture comprises contacting the fragments with anagent that binds to the detectably labeled starch fragments.
 18. Themethod of claim 17, wherein the agent is a lectin.
 19. The method ofclaim 17, wherein the agent is an antibody.
 20. The method of claim 1,wherein the sample is an aqueous slurry. 21-24. (canceled)
 25. A methodfor measuring α-amylase activity in a sample, comprising forming areaction mixture by contacting a sample with a detectably labeled starchsubstrate attached to a surface, for a time sufficient for α-amylase inthe sample to hydrolyze the starch substrate, thereby releasing solubledetectably labeled starch fragments, separating the soluble detectablylabeled starch fragments from the reaction mixture, and determining thelevel of hydrolysis of the detectably labeled starch substrate as ameasurement of α-amylase activity in the sample. 26-51. (canceled)
 52. Akit for measuring α-amylase activity in a sample, comprising a firstcontainer containing a detectably labeled starch substrate, a secondcontainer containing an α-amylase standard, and instructions formeasuring the α-amylase activity in the sample. 53-73. (canceled)
 74. Amethod of determining amylase in a sample comprising: placing about 6 mlincubation buffer in a substrate tube, warming the substrate tube to 45°C., adding about 200 mg of the sample to the warmed substrate tube,incubating the sample mixture in the substrate tube 10 min at 45° C.,adding about 4 ml stop buffer to the sample mixture in the substratetube, filtering the stopped sample mixture into a container, determiningthe fluorescence in the filtrate, and optionally converting thefluorescence value into a Falling Number Equivalent value. 75-79.(canceled)
 80. A method of determining amylase in a sample comprising:placing an about 3 g sample into a first container, adding a sufficientamount of fungal incubation buffer to have the total weight of sampleplus buffer equal of about 30 g, mixing the solution, extracting thesolution for 5 minutes at 45° C., adding the about 8 ml of the extractto a substrate tube, incubating extract in substrate tube 10 minutes at45° C., adding about 2 ml stop buffer the tube, mixing the contents ofthe tube, filtering the mixture into a second container, determining thefluorescence in the filtrate, and optionally converting the fluorescencevalue into an Enzyme Units Equivalent value. 81-87. (canceled)
 88. Amethod of determining amylase in a sample comprising: placing about 200mg of the sample into a container, adding about 20 ml fungal incubationbuffer to the sample, mixing the sample solution, diluting about 2 ml ofthe solution with 10 ml incubation buffer in a container, optionallyfurther diluting the diluted solution to obtain a concentration withinrange of about 0.1-1.0 SKB unit/ml, placing about 8 ml of the dilutedsample into a container, incubating the about 8 ml diluted sample 10minutes at 45° C., adding about 2 ml stop buffer to the 8 ml dilutedsample, filtering the mixture through a filter into a detectioncontainer, determining the fluorescence in the filtrate, and optionallyconverting the fluorescence value into an Enzyme Units Equivalent valueand multiplying by the dilution factor as a measure of the originalamylase concentration. 89-95. (canceled)